Because genetic polymorphism analysis has important application values in the fields of biomedical research and clinical practice, various techniques have been established to determine genetic polymorphisms. Some of the classical techniques include Restriction Fragment Length Polymorphism (RFLP), Single Strand Conformation Polymorphism (SSCP), Sequence Based Typing (SBT), Denaturing High Performance Liquid Chromatography (DHPLC), Allele-Specific PCR (ASPCR), Sequence Specific Oligonucleotide Probe (SSOP), etc. However, most of these methods have some shortcomings, such as high cost, low accuracy, or complicated procedures, etc. A common problem faced by these techniques is that neither is capable of high-throughput/large-scale genetic polymorphism analysis. In order to deal with the need for high-throughput analysis for genetic polymorphisms, new techniques are being developed by international efforts. For example, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), Ligase Detection Reaction (LDR), Single-Base Chain Extension (SBE), Bead Array, Universal Array, and High-Density Microarray, etc.
Among the above-mentioned techniques, Allele-Specific PCR (ASPCR) is an easy-to-practice method for polymorphism analysis. It was established by Newton et al. in 1989 (Newton, C. R., et al., Nucleic Acid Res. (1989) 17:2503-2516). Developed based on the PCR technique, ASPCR is also known as Amplification Refractory Mutation System (ARMS) or PCR-Sequence Specific Primer (PCR-SSP), etc. In order to analyze known mutations or polymorphisms in genetic sequences, ASPCR uses DNA polymerases without the 3′-5′ exonuclease activity so that if the 3′ end of a primer does not match the template, the primer can not be elongated and the PCR reaction is blocked. The ASPCR method is easy to practice but low-throughput, and is especially laborious when determining multiple polymorphisms simultaneously. In order to increase throughput, researchers have developed multiple strategies. Utilizing the theory of multiplex PCR, Robert et al. performed amplification of multiple polymorphic loci in which two separate PCR reactions were performed using primers specific for the wild-type and mutant alleles. See Ferrie, R. M., et al., Am. J. Hum. Genet. (1992) 51:251-262. PCR products are separated by electrophoresis in two lanes, one for wild-type and one for mutant, while different target loci are distinguished by the size of the PCR products. Gómez-Llorente et al. combined single reaction-multiplex PCR with capillary electrophoresis (Gómez-Llorente, M. A., et al., Early Hum. Dev. (2001) 65:S161-S164). Different target loci are distinguished by the size of PCR products while wild-type and mutant alleles are distinguished by labeling allele-specific primers with distinctly colored fluorescent dyes. Boniotto et al. combined single-reaction-multiplex PCR with melting temperature analysis to achieve multiplex polymorphism analysis (Boniotto, M., et al., J Immunol. Methods (2005) 304:184-188). They added GC tails to allele-specific primers in order to distinguish the Tm values of the two alleles and used SYBR Green I for quantitative fluorescent analysis. Eaker et al. combined ARMS with DNA Chip analysis by following multiplex ARMS amplification with hybridizing the labeled PCR products to DNA Chip with allele-specific oligonucleotides in order to discriminate polymorphisms (Eaker, S., et al., Biosensors Bioelectronics (2005) 21:933-939).
Universal array is a high-throughput technique for sequence analysis which was first developed by Barany et al. (U.S. Pat. No. 6,506,594). It combines the LDR with microarray in order to detect low abundance genetic point-mutations with high sensitivity. The 3′-end of the LDR common probe is labeled with fluorescent dye, while the 5′-ends of the allele-specific probes are linked to distinct cZip-code sequences. The cZip-code sequences are artificially designed and subject to critical filtering so that they are complementary to the Zip-code sequences on the universal array. Each combination of cZip-code and Zip-code corresponds to an allele of a mutation or single nucleotide polymorphism (SNP) in the target gene. The upstream and downstream probes are ligated by ligase when the allele-specific probe is complementary to the DNA target. The ligated products are used to hybridize with the universal array and sequence variation can be interpreted by analyzing the position of the Zip-code and the fluorescence signal intensity. This method has high sensitivity and is capable of accurate detection of 1% or less mutant SNP occurrence among wild-type sequences. When the cZip-code sequences are linked to other specific probes, the same array designed for one set of targets can be used for any target sequences, which makes the array universal. Combining universal array with liquid enzyme-catalyzed reaction greatly overcame the problem of low-specificity of allele-specific oligonucleotide arrays for the analysis of genetic polymorphism (or mutation). A similar method for determining the genotype of one or more individuals at a polymorphic locus employing amplification of a region of DNA using primers containing tags and hybridization of the products to one or more probes on a solid support was introduced by Affymetrix, Inc. (PCT Publication No. WO 01/29259).
There are about 20 million deaf patients who make up the largest handicap population in China. Approximately 50% of these cases are hereditary. Mutations in many different genes may cause hereditary hearing loss. The highly heterogeneous nature of this disorder led to genetic sequencing being the major clinical assay for deaf patients which is complicated to operate, low-throughput, and expensive. Thus there exists the need for a high-throughput and cost-effective method for genetic diagnosis to improve clinical management of the genetic information of hereditary deaf patients.
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